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Experimental Neurology 182 (2003) 288 –299
www.elsevier.com/locate/yexnr
Transplantation of porcine umbilical cord matrix cells into the rat brain
M.L. Weiss,a,* K.E. Mitchell,a J.E. Hix,a S. Medicetty,a S.Z. El-Zarkouny,b
D. Grieger,b and D.L. Troyera
a
Department of Anatomy and Physiology, Kansas State University, College of Veterinary Medicine, Manhattan, KS 66506-5602, USA
b
Department of Animal Science and Industry, Kansas State University, Manhattan, KS 66506-5602, USA
Received 1 August 2002; revised 28 October 2002; accepted 4 February 2003
Abstract
Immune rejection of transplanted material is a potential complication of organ donation. In response to tissue transplantation, immune
rejection has two components: a host defense directed against the grafted tissue and an immune response from the grafted tissue against the
host (graft vs host disease). To treat immune rejection, transplant recipients are typically put on immunosuppression therapy. Complications
may arise from immune suppression or from secondary effects of immunosuppression drugs. Our preliminary work indicated that stem cells
may be xenotransplanted without immunosuppression therapy. Here, we investigated the survival of pig stem cells derived from umbilical
cord mucous connective tissue (UCM) after transplantation into rats. Our data demonstrate that UCM cells survive at least 6 weeks without
immune suppression of the host animals after transplantation into either the brain or the periphery. In the first experiment, UCM cells were
transplanted into the rat brain and recovered in that tissue 2– 6 weeks posttransplantation. At 4 weeks posttransplantation, the UCM cells
engrafted into the brain along the injection tract. The cells were small and roughly spherical. The transplanted cells were positively
immunostained using a pig-specific antibody for neuronal filament 70 (NF70). In contrast, 6 weeks posttransplantation, about 10% of the
UCM cells that were recovered had migrated away from the injection site into the region just ventral to the corpus callosum; these cells also
stained positively for NF70. In our second experiment, UCM cells that were engineered to constitutively express enhanced green fluorescent
protein (eGFP) were transplanted. These cells were recovered 2– 4 weeks after brain transplantation. Engrafted cells expressing eGFP and
positively staining for NF70 were recovered. This finding indicates a potential for gene therapy. In the third experiment, to determine
whether depositing the graft into the brain protected UCM cells from immune detection/clearance, UCM cells were injected into the tail vein
and/or the semitendinosis muscle in a group of animals. UCM cells were recovered from the muscle or within the kidney 3 weeks
posttransplantation. In control experiments, rat brains were injected with PKH 26-labeled UCM cells that had been lysed by repeated sonic
disruption. One and 2 weeks following injection, no PKH 26-labeled neurons or glia were observed. Taken together, these data indicate that
UCM cells can survive xenotransplantation and that a subset of the UCM cells respond to local signals to differentiate along a neural lineage.
© 2003 Elsevier Science (USA). All rights reserved.
Keywords: Stem cells; Umbilical cord mesenchyme; Neural differentiation; Graft vs host disease
Introduction
Dr. Gerald D. Fischbach put it nicely: “Stem cells that
can develop into a variety of different types of nerve cells
and glia would be extremely valuable in the therapy of acute
and chronic neurological disorders.” (Larsen, 1999). In the
* Corresponding author. Department of Anatomy and Physiology,
Kansas State University, Coles Hall 105, Manhattan, KS 66506-5602,
USA. Fax: ⫹1-785-532-4557.
E-mail address: [email protected] (M.L. Weiss).
CNS, neural stem cell populations have been identified as
ependymal cells (Johansson et al., 1999), subventricular
zone astrocytes (Doetsch et al., 1999), and cells in the
subgranular zone of the dentate gyrus (Ray et al., 1993).
Thus, harvesting neural stem cells from the human patient
cannot be easily done because the location and distribution
of the neural stem cells makes it difficult to harvest a
substantial population of these cells for in vitro expansion/
manipulation and subsequent autologous transplantation.
Therefore, a question remains: Where can we get the neural
progenitor cells for transplantation?
0014-4886/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0014-4886(03)00128-6
M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
Two potential sources of neural stem cells have been
identified: first, the “transdifferentiation” of adult stem cells
derived from other primordial lineages, such as those derived from adult bone marrow stromal (BMS) cells, adipose
tissue, and hematopoietic tissue. BMS cells, for example,
are pluripotent cells that can differentiate into bone, cartilage, fat, muscle, tendon, neurons, and many other tissues
(Pittenger et al., 1999; Prockop, 1997). There is evidence
that BMS cells transplanted into rats can “home” in on
pathology. For example, BMS cells transplanted into rats
with induced liver damage contribute to the formation of
new hepatic oval cells that further differentiate into hepatocytes and ductal epithelium (Peterson et al., 1999). BMS
cells home in on damaged muscle in irradiated mice as well
(Ferrari et al., 1998; Gussoni et al., 1999). Kopen et al.
(1999) showed that BMS cells injected intracerebroventricularly (ICV) migrate extensively and differentiate into
glial cells and cells that express neurofilament (a neuronspecific marker) in neonatal mice. Two labs reported that
after bone marrow hematopoietic cells were injected intravenously, they migrated and expressed neuronal and microglial antigens in CNS (Brazelton et al., 2000; Mezey et al.,
2000). In vitro adult rat and human BMS cells can be
induced to differentiate into neurons (Woodbury et al.,
2000). A potential drawback of using BMS cells for autologous transplantation may be the difficulty in obtaining
sufficient material from older, diseased patients to expand
and differentiate.
A second source of stem cells is from embryonic stem
(ES) cell lines. ES cells are pluripotent and immortal (Bain
et al., 1995; Brustle et al., 1999; Deacon et al., 1998; Okabe
et al., 1996). ES cells have been successfully induced to
express neural phenotypes (Lee et al., 2000; Spenger et al.,
1994, 1995; Studer et al., 1995, 1996; Yan et al., 2001).
Several potential problems exist with transplanting ES cells:
First, in a significant percentage of cases, ES cells form
teratomas when transplanted into rodent brain (Thomson et
al., 1998). Second, to avoid rejection of grafted material,
donor recipients are typically put on immunosupression
therapy. Serious complications can arise from immune suppression and from the secondary effects of the immunosuppressive drugs, such as cyclosporine A (Kaplan, 1998;
Sander et al., 1996). Third, a dilemma exists about the moral
cost/societal benefit of therapeutic use of human ES cells.
Here, we present a potential source of stem cells for neural
transplantation: cells derived from umbilical cord mesenchyme
(UCM), also known as Wharton’s jelly mesenchyme or mucous connective tissue. In a preliminary report (Mitchell et al.,
2003), we demonstrated that pig UCM cells are stem cells that
can be differentiated into neurons and glia, can be maintained
and grown indefinitely in vitro, and can be genetically manipulated to express exogenous genes. Here, pig UCM cells were
found in the brains of rats following transplantation without
immunosuppressive therapy and a population of the UCM cells
appear to differentiate into neurons and migrate from the injection site.
289
Methods
Stem cell culture
The culture method is elaborated in another article
(Mitchell et al., 2003). Briefly, pig umbilical cords were
aseptically collected from preterm fetuses (approximately
60-day) at slaughter. Umbilical arteries and vein were
stripped manually and discarded. The remaining tissue was
minced finely in a sterile container in DMEM media with an
antibiotic (Gentamycin, 20 ␮g/ml, GIBCO BRL) and an
antifungal agent (Amphotericin B 250 ␮g/␮l, Sigma). The
explants were transferred to six-well plates containing the
above media along with 20% fetal bovine serum (FBS) for
culture. The primary cultures were left undisturbed for
about 7 days to allow migration of cells from the explants,
and then refed. They were fed thereafter twice weekly and
passaged as necessary (cells passaged at 80 –90% confluency). These stem cell cultures have been maintained beyond 100 population doublings and continue to grow vigorously (see Fig. 1).
Enhanced green fluorescent protein (eGFP)-expressing
UCM cells
The UCM cells were modified by S.Z. El-Zarkouny and
D. Grieger. The Sleeping Beauty Transposon system (a
generous gift from P. Hackett) was modified as follows: The
plasmid containing the transposon pT/HygR-eGFP (also a
gift from Hackett lab) was used as the template to generate
a PCR product of the hygromycin resistance-eGFP insert.
The neomycin resistance gene from the original transposon
vector (pT/SVNeo) was removed using the blunt cutters
BsaB1 and Nac1, and the hygromycin R/eGFP PCR product
was ligated into the original vector. We cotransfected this
plasmid along with the pCMV-SB plasmid containing the
transposase gene driven by the CMV promoter using lipofection (Lipofectamine, BRL). Hygromycin was added to
the medium after 3 days at 200 or 250 ␮g/ml to select for
transfected cells and stable transfection was attained after 3
weeks in selection media. The eGFP-expressing UCM cells
were maintained in hygromycin containing medium for two
to three passages prior to transplantation.
Transplantation procedure
UCM cells that had been in culture for 17, 40, 57, 58, 60
passages were used for the transplantation experiments.
There were no apparent differences in the results that could
be attributed to using one passage or another. In some cases,
the UCM cells were labeled with the lipophilic dye PKH 26
red (Sigma, St. Louis, MO) prior to transplantation. PKH 26
is a nontoxic permanent fluorescent marker (Ashley et al.,
1993; Honig and Hume, 1989). To transplant, the preconfluent cells were lifted with trypsin (7– 8 min). The trypsin
was inactivated by the addition of an equal volume of
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M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
Fig. 1. Phase contrast micrograph of uninduced UCM cells in culture. Two cell types are obvious: flat, fibroblast-like layer that adheres to the substrate with
scattered small round cells (arrows). When the cells become confluent, rounded clusters of cells that float above the substrate start appearing (dotted circle).
The culture can be sustained by passaging either the clusters or the adherent cells without apparent differences.
DMEM and 20% fetal bovine serum. The cells from several
plates were pooled and the number of cells was estimated by
counting on a hemocytometer. The final concentration was
adjusted to about 1000 cells/␮l. The UCM cells were transplanted into anesthetized Lewis or Sprague–Dawley rats
(2% halothane in oxygen) centrally via stereotaxic injection
(approximately 10,000 cells in 10 ␮l) or into the periphery
via tail vein injection (approx. 106 cells in 0.5 ml flushed
with 0.5 ml sterile saline) and intramuscular injection (approx. 106 cells in 0.4 ml), or via intramuscular injection
alone (approx. 106 cells in 0.5 ml). For stereotaxic injection,
a glass micropipette was lowered into the striatum (Bregma
⫹0.5, Lateral 3.4; D-V 5.0) and a 1-␮l bolus of graft cells
delivered over 1 min. After a 1-min interval, the micropipette was raised approximately 200 ␮m and a second 1-␮l
injection made. In this way, multiple injections were distributed along an injection tract until the entire 10-␮l volume was delivered. In other cases, the animals had a guide
cannula implanted in a previous surgical session prior to
delivering the cells via an injection cannula (Plastics One).
Control transplants (sterile saline alone, Con rats) were
performed in age-matched animals. In a separate control
experiment, two rats were transplanted with PKH 26-labeled UCM cells that had been previously lysed by sonic
disruption in phosphate buffer saline (for approximately 1
min). The disruption of the cells was confirmed by flow
cytometry after exposure to sonic disruption and by plating
an aliquot of the disrupted cells in growth media. Control
rats and normal animals with no treatment served as specificity and background controls for immunocytochemistry.
All animal manipulations were conducted in accordance
with PHS and Society for Neuroscience guidelines and with
the prior approval of the IACUC.
Immunocytochemistry
Previously described methods for detection of a single
antigen were used (Fitch et al., 2000; Fitch and Weiss,
2000). Briefly, Equithesin-anesthetized rats were sacrificed
by transcardial perfusion with heparinized isotonic saline
rinse followed by 10% buffered neutral formalin. The brains
were removed, postfixed 2 h, and cryoprotected in 20%
sucrose. Frozen sections were cut at 30 – 40 ␮m coronally
and sections were collected into three sets of adjacent sections, one set consisting of every third serial section. One set
of sections was processed for IC and the adjacent sets were
held in reserve in a cryoprotectant solution (Watson et al.,
1986). Free-floating tissue sections were immunocytochemically stained for GFP (Chemicon), pig-specific neurofilament 70 (NF70) (Chemicon), neuron-specific ␤-tubulin
(TuJ1) (Covalence Research Products), NFM (Chemicon),
and 2⬘, 3⬘-cyclic nucleotide-3⬘-phosphodiesterase (CNPase)
(Chemicon) and localized either with immunofluorescence
or with peroxidase using a commercially available ABC kit
(VectaStain). The monoclonal NF70 antibody was particularly valuable because it does not recognize rodent neurofilaments. When using this antibody, it was necessary to
substitute previously adsorbed secondary antibody (adsorbed for rat antigens, Jackson Labs) for the secondary
provided in the Vectastain kit. The tissue was incubated in
the following reagents: endogenous peroxidase elimination,
5% blocking serum, followed by the primary anti-serum,
fluorophore-labeled (Jackson Immuno, fluoroscein isothiocyanate or Molecular Probes, Alexafluor 480) or biotinlabeled (from the VectaStain kit) secondary antibody. The
tissue was triple rinsed with PBS– 0.2% Triton X-100 between each incubation. For immunofluorescence detection,
M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
the tissue were mounted on gelatin-chromium potassium
sulfate-coated microscope slides and air-dried. The stained
sections were examined using epifluorescence microscopy
after clearing and coverslipping with glycerol containing
N-propyl gallate to prevent fading. For immunoperoxidase
detection, the antigen was localized with diaminobenzidine
(DAB, Sigma) and hydrogen peroxide. Immunocytochemical (IC) labeling was considered positive if the signal was
distinctly above background and the signal was above that
seen in the negative controls (omission of primary antibody,
or labeling found in control or normal rats). To be considered double-labeled, the morphology and location of the cell
must appear identical in both brightfield (DAB) and fluorescence (PKH 26 or eGFP).
Results
Pig UCM cells in culture
Details about the cell culture, propagation and maintenance of pig UCM cells can be found in Mitchell et al.,
(2003). Briefly, when UCM cells initially grow outward
from explants two morphologically distinct populations of
cells are present: spherical or flat mesenchymal cells. When
the cells become confluent, they form spherical colonies that
remain attached to cells below (see Fig. 1). These colonies
resemble “neurospheres” (Reynolds and Weiss, 1992).
UCM cell culture can be maintained either by harvesting the
neurosphere-like cell clusters or by passage of preconfluent
flat and spherical cells without apparent differences. We
have maintained UCM cell cultures for more than 100
population doublings and they continue to grow vigorously.
Three cellular characteristics indicate that undifferentiated
UCM cells are a type of stem cell: (1) the number of
passages that they have been maintained in culture; (2) the
fact that UCM cells are telomerase-positive; and (3) UCM
cells make the receptor for stem cell factor, c-kit (data found
in Mitchell et al., 2003). UCM cells have been characterized
in vitro by immunocytochemistry and Western blotting
(Mitchell et al., 2003). UCM cells can be induced to differentiate into neurons and glia following the procedure described by Woodbury et al. (2000) (see Fig. 2, further details
in Mitchell et al., 2003). A small percentage of untreated
UCM cells and a larger percentage of differentiated UCM
cells exhibit positive staining for neural proteins. Within 1 h
of induction treatment, multiple “neurites” can be seen
extending from many cells, and the cell bodies become
rounded and refractile in phase contrast (Mitchell et al.,
2003). Fig. 2 shows how UCM cells respond to induction
using the Woodbury et al. protocol and examples of differentiated UCM cells that are IC positive for TuJ1, tau, and
NFM (see Mitchell et al., 2003, for full details). Here, we
transplanted undifferentiated, preconfluent pig UCM cells
into adult rats.
291
Experiment 1. Transplantation of pig UCM cells into rat
brain
Two injection methods were used with differing results.
The Hamilton syringe delivery method produced much less
tissue damage and a more discrete graft compared to the
animals that received the guide cannula implantation and
subsequent grafting via a secondary injection cannula. In
addition, the injection through the injection cannula was
more damaging to the UCM cells than the microliter syringe
(data not shown). In the animals injected with the Hamilton
syringe, the graft cells were located along the injection tract
and no gross brain damage was found 4 weeks after injection despite the large volume (4 ␮l) and many UCM cells
were found along the injection tract (see Fig. 3). In contrast,
the guide cannula animals had more extensive damage to
the brain associated with the larger diameter guide cannula
(data not shown). Apparently the brain tissue adjacent to
the implanted cannula had withdrawn slightly because
the transplanted cells were found distributed adjacent to the
guide cannula tract, as well as at the tip. Following recovery
from surgery, no complications were observed. No animals
died subsequent to transplantation and no unusual behaviors
were noted. There was no sign of brain tumor or teratoma,
immunological response, or glioma in transplant recipients;
all animals increased body weight. Four weeks after transplantation, there was no apparent glial scar. Large injection
volumes and guide cannula implantation caused obvious
tissue damage, as one would expect. Multiple nuclei, indicative of fusion with host cells, were not observed in the
transplanted cells and there was no evidence of uncontrolled
replication of UCM cells after transplantation.
After tissue processing, pig UCM cells were identified
either by PKH 26 fluorescent staining of dye-loaded cells or
by pig-specific NF70 immunocytochemical staining (see
Fig. 3–5). PKH 26 fluorescent staining was found throughout the cytoplasm and membrane. NF70 immunocytochemical staining was spread throughout the cell cytoplasm (see
Fig. 3–5). No NF70 or PKH 26 staining was found in
control animals. There was no evidence of immunological
response in the 2- to 8-week period after grafting; i.e., there
was no perivascular cuffing, no extracellular debris, and no
phagocytosis. A subset of the UCM cells had migrated into
the parenchyma of the brain away from the injection site. At
2– 4 weeks posttransplantation, most UCM cells appeared
as simple spherical cells 10 –15 ␮m in diameter with a
granulated cytoplasm. A small subset of the UCM cells had
single short processes extending from the cell body at this
time. Posttransplantation, many UCM cells were found
along the injection tract (see Fig. 3).
At 6 weeks, UCM cells were also found ipsilateral to the
transplantation site adjacent to the corpus callosum. Thus, a
subset of UCM cells had apparently migrated from the
injection site into the parenchyma (data not shown).
At 2– 6 weeks posttransplantation, a subset of the PKH
26-labeled UCM cells were immunocytochemically stained
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M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
Fig. 2. UCM cells induced to a neural phenotype in culture. (A–C) Change in cell morphology is shown in sequential photographs of a culture that was
induced to differentiate along the neural lineage (see text). (A) Uninduced UCM cells. (B) Same culture of cells after exposure to the full-term induction (FI)
protocol. (C) Same cells after 10 days in the long-term induction media (LTI). (FI and LTI protocols described in detail in Woodbury et al. (2000). Inset:
Note long processes and phase-bright cell body. The neurite-like short processes (arrows) and the growth cone-like projection at the distal end of a long
process. (D–F) Differentiated UCM cells demonstrate positive IC staining for neural markers: class III neuron-specific ␤-tubulin (TuJ1) (D), neurofilament
medium (NF-M) (E), or a neuron-specific microtubule-associated protein, tau (F). (G) High-power phase-contrast micrograph of cell exposed to LTI protocol.
Note: The granular material that resembles Nissl substance and the “neurites” with primary and secondary processes.
M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
293
Fig. 3. UCMs 2 weeks after transplantation. The same field is shown on the top and bottom. Left panels: Transplanted cells were identified by the PKH 26
dye loading (top) or by the expression of pig-specific NF70 immunocytochemical staining (bottom). The enclosed area is shown at higher magnification on
the right. Right panels: The top panel shows the relatively simple morphology of the UCMs 2 weeks after transplantation into the rat brain. For the most part,
the cells lack processes, have a granular cytoplasm, and stain brightly with the PKH 26 dye. The bottom panel shows immunocytochemical staining for
pig-specific NF70. The arrows indicate examples of cells that are both PKH 26-stained and positively immunocytochemically stained for NF70. The circles
indicate UCMs cells that do not stain for NF70, suggesting that not all UCMs differentiate along the neural lineage following transplantation. The asterisks
indicate cells that positively immunocytochemically stain for NF70 (bottom), but do not stain with PKH 26. The interpretation of this result is that the PKH
26 dye loading did not stain 100% of the UCMs.
for neural markers such as TuJ1 and MAP2 (see Fig. 4).
Positive staining for CNPase in some PKH 26 cells was also
detected, suggesting that some of the UCM cells may differentiate into oligodendrocytes (data not shown). It was
interesting to note that TuJ1, CNPase, and MAP2 staining
was found in PKH 26-negative cells that may not be part of
the grafted material (indicated by asterisks in Fig. 4).
Experiment 2. Transplantation of eGFP-expressing UCM
cells into the brain
At 4 weeks posttransplantation, eGFP-expressing UCM
cells were detected in the brain spread along the cannula
tract. The cytoplasm of these cells had a granular appearance and a large percentage of the cells stained positively
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M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
Fig. 4. Transplanted UCMs express neural-specific markers in rat brain. (A) Left: Pig UCMs, indicated by PKH 26 staining, 4 weeks after transplantation
into rat brain. Note that the PKH 26 staining is usually confined to the cell bodies. Right: Identical field as shown in the left panel. TuJ1 immunocytochemical
staining. UCMs that stain for the neural-specific marker TuJ1 are indicated by arrows. Arrowheads indicate PKH-labeled fibers that stain positively for TuJ1.
(A) Asterisks indicate PKH 26-positive cells that do not stain for TuJ1. This may indicate that not all graft cells differentiate along the neural lineage. (B)
Left: Pig UCMs 4 weeks after transplantation. Right: IC staining for the ␤-III tubulin protein (a neuronal marker), TuJ1. The filled circles indicate the large
number of double-labeled cells. (B) Asterisks indicate TuJ1-stained cells in the graft that may not originate from the graft (lack PKH 26 staining). This would
suggest that the graft may stimulate endogenous stem cell migration and differentiation. The arrowheads indicate the location of TuJ1 IC-positive fibers. (C)
M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
295
Fig. 5. Pig UCM cells were injected into the periphery. In this case, the UCM cells were delivered intramuscularly into the semitendinosis and intravenously.
Left: PHK 26-labeled cells are found along the im injection tract 4 weeks after injection. Right: PKH 26-labeled cells were found within the parenchyma
of the kidney. Finding transplanted UCM cells 4 weeks after injection suggests that the immune system does not clear these cells from the body.
for NF70 (see Fig. 4). The graft cells can be identified by
eGFP fluorescence or by using an anti-GFP antibody 2– 8
weeks posttransplantation (data not shown). With either
detection method, GFP staining found throughout the cell
cytoplasm and thus the morphology of the grafted cells was
revealed. The morphology of GFP-stained cells was simple
spherical or fusiform with zero, one, or two processes. The
exogenous nature of the eGFP-expressing cells was confirmed by double staining for pig-specific NF70 (see Fig. 4).
Extracellular GFP staining was never observed. There was
no evidence of phagocytosis or extracellular debris in the 2to 8-week survival period. In control animals, no eGFP
staining was observed around the injection site.
living cells were found in vitro. An aliquot containing
10,000 lysed cells was injected into two rats. One rat survived 1 week; the other survived 2 weeks after injection
prior to sacrifice and tissue processing. In both cases, cellular debris or red blood cells were found along the injection
tract (see Fig. 6B). No fluorescent labeling was found within
neurons or glia. On occasion, fluorescent blood cells were
observed along the injection tract (see arrows in Fig. 6B);
the red blood cells were easily distinguished from the PKH
26-labeled UCM cells by their smaller size and smooth,
round, or doughnut-like appearance.
Discussion
Experiment 3. Peripheral injection of pig UCM cells
UCM cells were injected into the periphery, intramuscularly (N ⫽ 3) or both intramuscularly (IM) and intravenously (IV) (N ⫽ 1), in a group of rats. Three weeks after
IM injection, PKH 26-labeled UCM cells were recovered
from the injection site (see Fig. 5). Three weeks after IM
and IV injection, PKH 26-labeled UCM cells were engrafted in the parenchyma of the kidney (see Fig. 6). No IC
characterization was performed in these cases.
Experiment 4. Brain injection of disrupted pig UMC cells
Flow cytometry indicated that sonic disruption fragmented the cells prior to transplantation (see Fig. 6A). No
Here, we present four lines of evidence indicating that
pig UCM cells are stem cells that do not stimulate immune
rejection when transplanted into the adult rat. First, pig
UCM cells survive 2– 6 weeks after transplantation into the
rat without immune suppression therapy. Second, pig UCM
cells respond to differentiation cues and modify their morphology and neurochemical phenotype to resemble neural
cells both in cell culture and after transplantation into the rat
brain. At 2 and 4 weeks after injection, most pig UCM cells
found in the rat brain were simple spherical cells with a
granular cytoplasm. At 6 weeks, some UCM cells had
migrated from the injection site to a site adjacent to the
corpus callosum and had short processes. Third, pig UCM
cells that were injected into the periphery were recovered in
Same field is shown on the left and right. Left panel: engrafted UCM cells. Right panel: IC staining for neuron-specific microtubule-associated protein 2
(MAP2). The filled circles indicate the double-labeled cells. The asterisks indicate MAP2-stained cells that may not be of graft origin (they lack PKH 26).
The arrowheads indicate MAP2 IC-positive fibers. (D) Left: UCM cells that were engineered to express eGFP were detected 4 weeks after transplantation.
Note that most of the cells have a granular cytoplasm and a few have short primary processes. Right: Many of the eGFP-expressing cells also stain for
pig-specific NF70, confirming that they are from porcine origin. The filled circles indicate corresponding areas in both fields. There was a large percentage
of double-labeled cells.
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M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
Fig. 6. Previously disrupted PKH26-labeled UCM cells do not label neurons or glia in rat brain following transplantation. (A) Flow cytometry was used to
assess the fragmentation of pig UCM cells following sonic disruption. Left: UCM cells prior to disruption (Control). Middle: Flow cytometry data following
sonic disruption indicates that 0.3% of the population remains within the gated region. Right: Following a second round of sonic disruption, 0.2% of the
population remains within the gated region. Culturing of an aliquot of the lysate did not yield cells. Taken together, these data indicate that the UCM cells
were destroyed prior to transplantation. (B) Top: One week after injection of disrupted PKH26-labeled cells, the area along the injection track was examined.
While the background fluorescence was higher along the injection track, red blood cells were the only fluorescent cells found in this area (indicated by the
arrowheads). No fluorescent neurons or glia were observed. Bottom: In contrast, when intact PKH 26-labeled UCM cells are injected, fluorescent cells were
recovered in and around the injection tract 2– 6 weeks following injection (data from a 4-week survival postinjection is shown here). Note that the fluorescent
graft cells (indicated by triangles in bottom panel) are larger and more irregular in appearance than the small, doughnut-shaped red blood cells indicated in
the top panel. Calibration bar ⫽ 20 ␮m.
the injection site and the kidney 3 weeks later. Fourth, after
injection of disrupted UCM cells, no cells labeled by PKH
26 were found. Together these results indicate that pig UCM
cells are relatively nonimmunogenic, that they respond to
local cues found in the adult rat, and that these cells engraft
without stimulating significant immune rejection.
M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
The properties of UCM cells in culture
While umbilical cord blood banking has become a reality
with identified clinical significance (Huhn, 2000; Jacobs
and Falkenburg, 1998; Nishihira et al., 1998; Rendine et al.,
2000; Wagner and Kurtzberg, 1998), the loose connective
tissue matrix of the umbilical cord, e.g., the Wharton’s Jelly
matrix, is not widely appreciated as a source of stem cells.
Eyden et al. and McElreavey et al. recognized myofibroblast-like cells in the umbilical cord matrix (Eyden et al.,
1994; McElreavey et al., 1991), but apparently that work
was not pursued. We speculated and subsequently demonstrated that the umbilical cord Wharton’s Jelly is a rich
source of primitive mesenchymal stem cells (Mitchell et al.,
2003). Pig UCM cells appear poised to differentiate into
neurons and are rapidly induced along this pathway and
express neural markers such as TuJ1, NF-M, and tau in vitro
(for further characterization, see Mitchell et al., 2003).
Thus, we were interested to determine the fate of pig UCM
cells after transplantation into adult rat brain. It is interesting
to speculate that stem cells derived from UCM may serve as
the source of the persistent fetal cells found in the mother
(Bianchi, 1996). It is possible that some special property of
the UCM stem cells may permit them to evade the host
immune system.
Here, pig UCM cells were isolated from Wharton’s jelly,
the matrix of the umbilical cord, rather than cord blood.
Because umbilical cord blood has been found to be a source
of hematopoietic stem cells (Broxmeyer et al., 1989, 1990;
Gluckman, 1996; Lu and Ende, 1997), we confirmed that
the UCM cells used here are not derived from cord blood.
Umbilical blood vessels were stripped from the cord before
explant preparation, and the UCM cells tested negative for
markers of the hematopoietic lineage (such as CD34 and
CD45; Weiss et al., unpublished observations).
Transplantation and recovery of UCM cells
After tissue processing, the transplanted cells were identified in three different ways. First, the UCM cells that were
loaded with PKH 26 prior to transplantation were recovered
by observing fluorescent cells, and not fluorescent debris,
along the injection track and elsewhere in the brain. Red
blood cells (RBCs) also fluoresce and were found in the
brains of transplanted and control animals but RBCs were
easily differentiated from the transplanted UCM cells based
upon their size and shape (see Fig. 6). Injection of disrupted
PKH 26-labeled UCM cells did not label host neurons or
glia. This indicates that the lysed PKH 26-labeled UCM
cells do not stain host cells following phagocytosis, possibly
due to enzymatic degradation of the dye. Second, many, but
not all, transplanted UCM cells were identified by their
staining for the pig-specific NF70 IC staining 2– 6 weeks
after introduction. The NF70 antibody does not recognize
rodent epitopes, and in these experiments NF70 IC staining
was never found in normal or control animals. In contrast,
297
NF70 staining was often colocalized with PKH 26 fluorescence. Most important, NF70 staining was not found in
debris, phagocytic cells, or lysozomal vesicles. Third, the
UCM cells that were engineered to produce eGFP prior to
transplantation were recovered either by observing eGFP
using epifluorescence or by IC staining for the GFP protein
and immunoperoxidase. IC staining for GFP would not be
expected in the case of UCM cell lysis because the released
GFP protein and mRNA is likely to be degraded by phagocytic cells. In both cases, eGFP was found in transplanted
animals, but not in either control group. All three of these
recovery methods indicated that the transplanted cells were
found in the rat brain 2– 6 weeks after injection. Further, our
results indicated that the transplanted cells do not form
tumors. We cannot at this time address whether the UCM
cells replicate after transplantation. Ongoing work involving the transplantation of 150 eGFP-expressing UCM cells
and a time series analysis indicates that UCM cells may
replicate between transplantation and a 2- to 8-week recovery period (Medicetty et al., in preparation).
While we did not directly evaluate the infiltration of
lymphocytes, macrophages, natural killer cells, microglia,
or astrooytes, our findings suggest that the grafted cells were
not recognized or attacked. There was no indication of
cellular lysis or cellular debris in animals transplanted with
UCM cells and there were no obvious signs of immunological response, such as infiltration of immune cells, perivascular cuffing, extracellular debris, or graft antigens within
cells with glial morphology or size. In contrast, when lysed
cells were injected into the brain, debris and RBCs were
found in the injection site. Further, because UCM cells were
recovered in the kidney 3 weeks after peripheral injection or
up to 6 weeks after injection into the brain, we believe that
UCM cells avoid immune surveillance. These findings are
in contrast to previous findings by Larsson et al., who
transplanted fetal pig ventral mesencephalic tissue (E27 or
E29) into rat brain and demonstrated immune rejection in
2– 4 weeks (Larsson et al., 2000). There are several possible
explanations for these disparate results. One possible explanation is that primitive undifferentiated stem cells do not
have the full complement of surface antigens, and thus do
not stimulate immune rejection. This explanation is in
agreement with recent work performed on human ES cells
which indicated that the expression of MHC class I proteins
correlated with the differentiation of ES cells (Drukker et
al., 2002). A second explanation may be that extraembryonic tissues, such as the umbilical cord, have some endogenous factors that suppress immune recognition, and stem
cells derived from this tissue may be immunosuppressive, as
discussed above. Previous work had indicated a significant
reduction in the immune response to expanded neural precursor cells compared with primary fetal tissue suspensions
(Armstrong et al., 2001). In Armstrong et al.’s study, small
but significant levels of porcine major histocompatibility
complex (MHC) expression were found in the primary tissue suspensions and no expression was found in expanded
298
M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299
neural precursor cells. From the present results, it is unknown whether MHC expression or lack thereof accounts
for the survival of the transplanted material.
limiting factor for successful grafting. Pig UCM cells appear to overcome this limitation and, thus, may prove to be
therapeutically useful in the future.
Fate of transplanted tissue
Acknowledgments
Implanted UCM cells primarily developed into neural
grafts as indicated by IC staining for pig-specific NF70 at
2– 6 weeks after transplantation and by the IC staining for
other neuron-specific markers weeks after transplantation.
For example, positive double-labeling of UCM cells for two
other cytoskeletal markers also identified the transplanted
cells as neurons: class III TuJ1, and microtubule-associated
protein 2. In contrast, fewer oligodendrocytes originated
from the grafted material, as indicated by the few cells that
double-stained for CNPase and PKH 26. It was noted that
associated with the grafted tissue, pig-specific NF70-stained
cells were found that did not contain PHK 26 (these cells are
indicated by asterisks in Fig. 3 and 4). We interpret this
result as indicating that not all of the UCM cells were
labeled in vitro prior to transplantation. The observed immunocytochemical staining of UCM cells shown here extends our previous studies of in vitro cells that had indicated
that UCM cells could be induced to differentiate into neurons and glia (see Fig. 2 and Mitchell et al., 2003).
Therapeutic potential of UCM cells
In Parkinson’s disease (PD), human fetal mesencephalic
transplants were once used as the source of replacement
cells, but moral/ethical concerns associated with the use of
fetal tissue and the scarcity of the tissue and other problems
associated with obtaining enough tissues have been barriers
to the widespread use of transplantation. Currently, ventral
mesencephalic cells obtained from fetal pigs are used for
xenotransplants in PD patients (Bjorklund and Lindvall,
2000; Deacon et al., 1997). Xenotransplants have problems
such as graft vs host disease or immune rejection (Larsson
et al., 2000). For therapeutically useful numbers of the
xenografted cells to survive, the host’s immune system must
be suppressed (Deacon et al., 1997). Despite immune suppression, it was estimated that about 5–10% (Bjorklund,
1991) or about 25% (Brundin et al., 2000). of the harvested
cells survive transplantation. We have begun to analyze
human UCM stem cells and found that they can be induced
to differentiate along the neural lineage in vitro (Medicetty,
et al., unpublished observations), just as we have shown for
pig UCM cells (see Fig. 2 and Mitchell et al., 2003). Currently, we are investigating whether human UCM cells lack
cell surface antigens (MHC class I and II proteins) and
whether the expression of those proteins change after differentiation along the neural lineage (Drukker et al., 2002).
This work, along with efforts to introduce clinically useful
genes (Mannes et al., 1998), is important to determining the
therapeutic potential of UCM cells. At present, overcoming
or reducing the immune responses of the host is a critical
Cameron Fahrenholtz, Lois Morales, Tammi Hildreth,
and Katrina Fox are thanked for technical assistance. Dr.
Duane Davis is thanked for reviewing an earlier draft of the
manuscript and providing ideas and moral support. This
work was supported, in part, by a KSU USRG award and by
NIH NS 34160 to M.L.W.; by NIH COBRE RR15563 to
K.E.M.; and by Project 481326 (DT) from the Kansas Agricultural Experiment Station.
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